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Thiol-NO adducts such as S-nitrosoglutathione (GSNO) are endogenous bronchodilators in human airways. Decreased airway S-nitrosothiol concentrations are associated with asthma. Nitric oxide (NO), a breakdown product of GSNO, is measured in exhaled breath as a biomarker in asthma, an elevated fraction of expired NO (FENO) is associated with asthmatic airway inflammation. We hypothesized that FENO could reflect airway S-nitrosothiol concentrations. To test this hypothesis, we first studied the relationship between mixed expired NO and airway S-nitrosothiols in patients endotracheally intubated for respiratory failure. The inverse (Lineweaver-Burke type) relationship suggested that expired NO could reflect the rate of pulmonary S-nitrosothiol breakdown. We thus studied NO evolution from the lungs of mice (GSNO reductase −/−) unable reductively to catabolize GSNO. More NO was produced from GSNO in the −/− compared to wild type lungs. Finally, we formally tested the hypothesis that airway GSNO increases FENO using an inhalational challenge model in normal human subjects. FENO increased in all subjects tested, with a median t1/2 of 32.0 min. Taken together, these data demonstrate that FENO reports, at least in part, GSNO breakdown in the lungs. Unlike GSNO, NO is not present in the lungs in physiologically relevant concentrations. However, FENO following a GSNO challenge could be a non-invasive test for airway GSNO catabolism.

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

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HIV infects long-lived CD4 memory T cells establishing a latent viral reservoir that necessitates lifelong anti-retroviral therapy (ART). How this reservoir is formed so swiftly remains unknown. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, which can drive recruitment of cells expressing the CCR2 receptor including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find 1) treatment of humanized mice with anti-CCL2 antibodies during HIV infection decreases reservoir seeding and 2) CCR2/5+ cells from the blood of HIV-infected individuals on long term ART contain significantly more provirus than CCR2/5-negative memory or naïve cells. Together, these studies support a model where the host’s innate inflammatory CCL2 response to HIV infection recruits CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation likely contributing to rapid formation of the latent HIV reservoir.GRAPHICAL ABSTRACTWhy is the latent HIV reservoir established so early following infection? An innate immune response occurs during acute infection that establishes a “zone of inflammation” (step 1). The CCL2 chemokine is produced in part through IFI16 sensing of HIV DNA in abortively infected cells. CCL2 promotes rapid recruitment of CCR2/5+ memory CD4 T cells (step 2). Many of these cells become productively infected (step 3) and a fraction become latently infected (step 4). Thus, HIV hijacks the host inflammatory response to rapidly establish the latent reservoir. In support of this model, we find HIV reservoir reduction in humanized mice treated with anti-CCL2 antibodies during early infection. Further, we find that CCR2/5+ CD4 T cells harbor a substantial fraction of detectable proviruses in the blood of HIV-infected individuals on long-term suppressive ART.Abstract Figure

An ability to activate latent human immunodeficiency virus type 1 (HIV-1) expression could benefit many HIV cure strategies; however, most latency-reversing agents (LRAs) have proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i’s), SB-216763 and tideglusib (the latter already in phase II clinical trials), that activate AKT/mTOR signaling were tested. These GSK-3i’s reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to simian immunodeficiency virus (SIV)-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined. IMPORTANCE If combined with immune therapeutics, latency-reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8(+) T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing novel LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme responsible for reverting protein S-nitrosylation (SNO). In this issue, Salerno et al. [1] provide evidence that GSNOR deficiency - and thus elevated protein S-nitrosylation - accelerates cardiomyocyte differentiation and maturation of induced pluripotent stem cells (iPSCs). GSNOR inhibition (GSNOR-/- iPSCs) expedites the epithelial-mesenchymal transition (EMT) and promotes cardiomyocyte progenitor cell proliferation, differentiation, and migration. These findings are consistent with emerging roles for protein S-nitrosylation in developmental biology (including cardiomyocyte development), aging/longevity, and cancer.

Interleukin 1β is a pro-inflammatory cytokine important for both normal immune responses and chronic inflammatory diseases. The regulation of the 31 kDa proIL-1β precursor coded by the IL1B gene has been extensively studied in myeloid cells, but not in lymphoid-derived CD4 T cells. Surprisingly, we found that some CD4 T cell subsets express higher levels of proIL-1β than unstimulated monocytes, despite relatively low IL1B mRNA levels. We observed a significant increase in IL1B transcription and translation in CD4 T cells upon ex vivo CD3/CD28 activation, and a similar elevation in the CCR5+ effector memory population compared to CCR5- T cells in vivo. The rapid and vigorous increase in IL1B gene transcription for stimulated monocytes has previously been associated with the presence of Spi-1/PU.1 (Spi1), a myeloid-lineage transcription factor, pre-bound to the promoter. In the case of CD4 T cells, this increase occurred despite the lack of detectable Spi1 at the IL1B promoter. Additionally, we found altered epigenetic regulation of the IL1B locus in CD3/CD28-activated CD4 T cells. Unlike monocytes, activated CD4 T cells possess bivalent H3K4me3+/H3K27me3+ nucleosome marks at the IL1B promoter, reflecting low transcriptional activity. These results support a model in which the IL1B gene in CD4 T cells is transcribed from a low-activity bivalent promoter independent of Spi1. Accumulated cytoplasmic proIL-1β may ultimately be cleaved to mature 17 kDa bioactive IL-1β, regulating T cell polarization and pathogenic chronic inflammation.

Human immunodeficiency virus type 2 (HIV-2) infection results in a milder course of disease and slower progression to AIDS than does HIV-1. We hypothesized that this difference may be due to degradation of the sterile alpha motif and HD domain 1 (SAMHD1) host restriction factor by the HIV-2 Vpx gene product, thereby diminishing abortive infection and pyroptotic cell death within bystander CD4 T cells. We have compared CD4 T cell death in tonsil-derived human lymphoid aggregate cultures (HLACs) infected with wild-type HIV-2, HIV-2 ΔVpx, or HIV-1. In contrast to our hypothesis, HIV-2, HIV-2 ΔVpx, and HIV-1 induced similar levels of bystander CD4 T cell death. In all cases, cell death was blocked by AMD3100, a CXCR4 entry inhibitor, but not by raltegravir, an integrase, indicating that only early life cycle events were required. Cell death was also blocked by a caspase-1 inhibitor, a key enzyme promoting pyroptosis, but not by a caspase-3 inhibitor, an important enzyme in apoptosis. HIV-1-induced abortive infection and pyroptotic cell death were also not reduced by forced encapsidation of HIV-2 Vpx into HIV-1 virions. Together, these findings indicate that HIV-2 and HIV-1 support similar levels of CD4 T cell depletion in vitro despite HIV-2 Vpx-mediated degradation of the SAMHD1 transcription factor. The milder disease course observed with HIV-2 infection likely stems from factors other than abortive infection and caspase-1-dependent pyroptosis in bystander CD4 T cells. IMPORTANCE CD4 T cell depletion during HIV-1 infection involves the demise of bystander CD4 T cells due to abortive infection, viral DNA sensing, inflammasome assembly, and death by caspase-1-dependent pyroptosis. HIV-2 infection is associated with milder disease and lower rates of CD4 T cell loss. We hypothesized that HIV-2 infection produces lower levels of pyroptosis due to the action of its Vpx gene product. Vpx degrades the SAMHD1 restriction factor, potentially reducing abortive forms of infection. However, in tonsil cell cultures, HIV-2, HIV-2 ΔVpx, and HIV-1 induced indistinguishable levels of pyroptosis. Forced encapsidation of Vpx into HIV-1 virions also did not reduce pyroptosis. Thus, SAMHD1 does not appear to play a key role in the induction of bystander cell pyroptosis. Additionally, the milder clinical course of HIV-2-induced disease is apparently not explained by a decrease in this inflammatory form of programmed cell death.

Seeding of the latent HIV reservoir occurs quickly within hours to a few days after initial viral infection. It is the persistence of virus within this reservoir that thwarts an HIV cure and necessitates life-long antiretroviral therapy (ART). Central memory CD4 T cells harboring latent HIV proviruses form a major part of this reservoir. How the virus establishes latency so rapidly within this subset of CD4 T cells remains unknown. We find that HIV infection leads to rapid production of the CCL2 chemokine in human lymphoid CD4 T cells. Cas9RNP-mediated depletion of IFI16 and STING genes significantly reduces production of CCL2 occurring in response to HIV. IFI16 was previously implicated as the key sensor triggering CD4 T cell pyroptosis. Thus, infection may also stimulate a second pathway of IFI16-mediated signaling involving STING culminating in CCL2 production. CCL2 functions a potent chemoattractant for cells expressing CCR2. Lymphoid CCR2+ CD4 T cells express the CCR5 co-receptor for HIV and markers of central memory. Additionally, these cells express many markers previously associated with the latent HIV reservoir. CCR2+ CD4 T cells are highly permissive to latent and productive infection by both R5 and X4-tropic viruses. When CCR2+ CD4 T cells are purified from HIV-infected individuals on suppressive ART, these cells are enriched up to 75-fold for HIV proviral DNA compared to naïve cells (n=10, p=0.0078). Together, our findings support a model where HIV infection triggers the production of CCL2, leading to directed recruitment and infection of CCR2+ CD4 central memory T cells. Thus, HIV hijacks a component of the innate immune response to rapidly recruit precisely those target cells that ensure its long term persistence.

HIV infection rapidly causes localized inflammation, which contributes to viral pathogenesis by recruiting cells to become infected. At the same time, the stable HIV reservoir forms swiftly and persists over the life of the individual, likely due to infection of long-lived central memory CD4 T cells. The elimination/control of the reservoir is critical to an HIV cure. We sought to understand how host inflammatory responses to HIV drive the formation of the viral reservoir. Our early studies found that CD4 T cells rapidly produce the chemoattractant CCL2 following HIV infection, and this host response is dependent on HIV reverse transcription but not integration of viral DNA. We proposed that CCL2 produced early in response to HIV lures cells bearing the receptor—CCR2—to the zone of infection. Using mass cytometry, we identified a novel population of human lymphoid CCR2+ CD4 T cells that co-express CCR5 and have a central memory phenotype, along with markers associated with the HIV reservoir. We found that CCR2/5+ cells migrate in response to CCL2, fuse to R5 and X4-tropic HIV and support productive and latent infection. To measure the HIV reservoir in vivo, we sorted CCR2/5+ cells from PBMCs of HIV-infected ART-suppressed individuals and measured integrated HIV proviral DNA. Our preliminary findings show that CCR2/5+ cells have significantly higher levels of integrated HIV than CCR-negative memory (12-fold, p = 0.046) or naïve cells (28-fold, p = 0.012). We propose that HIV hijacks the host response to the virus to recruit CCR2+ cells, which may become latently infected to form the long-lived reservoir, providing a link between inflammation and reservoir seeding. We are now testing if blockade of CCL2 disrupts formation of the HIV reservoir.